Olfactomedin 1 is a secreted glycoprotein belonging to a family of olfactomedin domain-containing proteins. Two zebrafish olfactomedin 1 genes (olfm1a and olfm1b) are expressed in developing retina and other neuronal tissues. We investigated the functions of Olfactomedin 1 in eye development. Over-expression of full-length Olfactomedin 1, as well as C-terminally truncated forms increased the thickness of the optic nerve and facilitated its branching in the optic tectum. In earlier stage embryos, such over-expression also altered the expression domains of eye-filed markers, rx2 and rx3. Injection of morpholino oligonucleotides against olfm1 reduced eye size, inhibited optic nerve extension, increased the number of apoptotic cells in the retinal ganglion cell and inner nuclear layers, and suppressed the separation of eyes from forebrain. Injection of RNAs encoding full length Olfactomedin 1 or truncated forms lacking the C-terminal olfactomedin domain restored normal eye size in morpholino oligonucleotide-injected embryos, while injection of RNA encoding the N-terminally truncated form of Olfactomedin 1 did not exhibit this rescue effect. The reduction of eye size by morpholino oligonucleotide injection was also rescued by a co-injection of runxb (but not by runxa) RNA encoding a transcription factor expressed in zebrafish neural tissues. The expressions of neurotrophin receptor, trkc, in the retina was slightly increased by olfm1 RNA injection and strongly decreased by morpholino oligonucleotide injection. Zebrafish olfm1s may play roles in early eye determination, differentiation, optic nerve extension and branching of ganglion cell axon terminals. The N-terminal region of Olfactomedin 1 is critical for these effects.